High-purity mogrosides and process for their purification

ABSTRACT

The present invention provides a process for preparation of highly purified mogrosides mixture from low purity mogrosides mixture. The process comprises providing a mixture of low purity mogrosides, dissolving the low purity mogrosides mixture in water or an aqueous alcohol solution to form an initial solution of mogrosides, passing the initial solution through a column system, wherein the column system comprises a plurality of columns, and each column is packed with a sorbent having different affinities to impurities and mogrosides so that one or more columns retains more mogrosides than other columns, washing the columns to remove impurities with an acidic aqueous solution, a basic aqueous solution, and an aqueous alcoholic solution successively, eluting the columns with an aqueous alcohol solution that contains higher alcohol content than the aqueous alcohol solution used in the washing step, wherein the eluate from the columns with high content of mogrosides are combined, and drying the combined eluate to obtain high purity mogrosides with the content of the total mogrosides are more than 70% (w/w). The present invention also provides a sweetener mixture and product comprising high purity mogrosides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/219,721 filed Aug. 29, 2011, which claims the benefit of USprovisional application entitled “High-Purity Mogrosides” filed Sep. 3,2010 having Ser. No. 61/379,729.

FIELD OF THE INVENTION

The invention relates to a process for purification of high puritymogrosides from Siraitia grosvenori fruit extract, particularly highpurity mogrosides mixtures with high mogroside V content, and further tohigh purity mogrosides and their applications in food and beverageproducts.

DESCRIPTION OF THE RELATED ART

Luo han guo generally refers to a fruit of Siraitia grosvenori, a memberof the Cucurbitaceae family, is a plant native to some regions ofsouthern Asia and China. The sweet taste of luo han guo mainly comesfrom triterpene glycosides generally known as mogrosides or mogrolglycosides. Mogrosides comprise only about 1% of the luo han guo fruit.There are a number of mogrosides identified in luo han guo but generallymogroside V (CAS No: 88901-36-4) has the highest concentration comparedto others (Table 1). Mogrol glycosides have the same coremolecule—mogrol or oxo-mogrol and differ from each other by number andtype of glycosidic residues bonded to mogrol or oxo-mogrol molecules.

TABLE 1 Mogrosides present in Luo han guo fruits Substance Mol. FormulaMol. Weight Mogroside IIE C₄₂H₇₂O₁₄ 801.01 Mogroside III C₄₈H₈₂O₁₉963.15 Mogroside IV C₅₄H₉₂O₂₄ 1125.29 Mogroside V C₆₀H₁₀₂O₂₉ 1287.43Mogroside VI C₆₆H₁₁₂O₃₄ 1449.58 11-oxo-Mogroside V C₆₀H₁₀₀O₂₉ 1285.42Siamenoside I C₅₄H₉₂O₂₄ 1125.29 Grosmomoside I C₅₄H₉₂O₂₄ 1125.29

Various extraction techniques are used to isolate mogrosides from luohan guo fruits. As a result luo han guo powdered extracts are beingprepared which usually contain 30-65% w/w of total mogrosides, andmogroside V content of those materials can vary as much as 18-55%. Suchextracts generally cannot be used as a sweetener in foods and beveragesas they contain substantial amount of impurities; some of the impuritiespossess undesirable organoleptic properties, thus affecting the color,flavor, odor and taste profile of luo han guo extract.

Non-limiting examples of such impurities include proteins, pigments,polysaccharides, aldehydes, unsaturated aldehydes, methyl ketones, butylcrotonate, and phenolic compounds.

Hence there is a need for a commercially viable process for enhancingmogrosides content, particularly mogroside V content from low purity luohan guo preparations and removing the undesirable impurities so as tosignificantly improve the organoleptic characteristics of luo han guopreparations and thus allowing their usage in food.

No techniques are currently available for purifying of low purity luohan guo extract into high purity mogrosides. Therefore, there is a needfor a process of for purification of high purity mogrosides fromSiraitia grosvenori fruit extract.

SUMMARY OF THE INVENTION

The invention relates to a process for preparation of high puritymixtures of mogrosides from Siraitia grosvenori fruit extract.

In one embodiment, the process for preparation of highly purifiedmogrosides mixture from low purity mogrosides mixture comprises:

a. providing a mixture of low purity mogrosides;

b. dissolving the low purity mogrosides mixture in water or an aqueousalcohol solution to form an initial solution of mogrosides;

c. passing the initial solution through a column system, wherein thecolumn system comprises a plurality of columns, and each column ispacked with a sorbent having different affinities to impurities andmogrosides so that one or more columns retains more mogrosides thanother columns;

d. washing the columns to remove impurities with an acidic aqueoussolution, a basic aqueous solution, and an aqueous alcoholic solutionsuccessively;

e. eluting the columns with an aqueous alcohol solution that containshigher alcohol content than the aqueous alcohol solution used in thewashing step, wherein the eluate from the columns with high content ofmogrosides are combined; and

f. drying the combined eluate to obtain high purity mogrosides with thecontent of the total mogrosides are more than 70% (w/w).

In another embodiment of the process, in the washing step (d), the waterto alcohol ratio (vol/vol) in the aqueous alcoholic solution is 99.9:0.1to 60:40, and wherein in the eluting step (e), the water to alcoholratio (vol/vol) of the aqueous alcoholic solution is 60:40 to 0.1:99.9.

In another embodiment of the process, in the washing step (d), theacidic aqueous solution comprises HCl, and the basic aqueous solutioncomprises NaOH.

In another embodiment of the process, the aqueous alcoholic solutioncomprises alcohol that is selected from the group consisting ofmethanol, ethanol, n-propanol, 2-propanol, 1-butanol, and 2-butanol.

In another embodiment of the process, the sorbent is a macroporouspolymeric adsorption resin capable of adsorbing mogrosides.

In another embodiment of the process, the plurality of columns areconsecutively connected.

In another embodiment of the process, the plurality of columns areconnected in parallel.

In another embodiment of the process, it further comprises:

g. dissolving the high purity mogrosides in water or an aqueousalcoholic solution to result in a solution of mogrosides;

h. passing the solutions of mogrosides through a sequence ofultrafiltration and/or nanofiltration membranes with MWCO size from 1000to 2500 to increase the purity of mogrosides mixture (total mogrosidescontent, % dry basis); and

i. drying the obtained mogrosides mixture solution to obtain highlypurified mogrosides mixture.

The present invention further provides a sweetener mixture comprisingthe high purity mogrosides; wherein the high purity mogrosides areblended with another high intensity sweetener.

In one embodiment of the sweetener mixture, the another high intensitysweetener is selected from the group consisting of steviol glycosidesincluding a purified sweet steviol glycoside mixture, stevioside,rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,rebaudioside E, rebaudioside F, dulcoside A, dulcoside B, rubusoside,and stevia; siamenoside; mogroside IV; mogroside V; Luo Han Guosweetener; monatin and its salts (monatin SS, RR, RS, SR); glycyrrhizicacid and its salts; curculin; thaumatin; monellin; mabinlin; brazzein;hernandulcin; phyllodulcin; glycyphyllin; phloridzin; trilobatin;baiyunoside; osladin; polypodoside A; pterocaryoside A; pterocaryosideB; mukurozioside; phlomisoside I; periandrin I; abrusoside A;cyclocarioside I; and combinations thereof.

The present invention also provides a product comprising the high puritymogrosides.

In one embodiment of the product, the product is selected from the groupconsisting of food, beverage, pharmaceutical composition, tobacco,nutraceutical, oral hygienic composition, or cosmetic.

One objective of the invention to develop an efficient method ofpreparation of highly purified mogro sides' mixture from low puritymogrosides' mixture.

Another objective of the invention is to develop a method of preparationof high mogroside V content luo han guo sweetener from low mogroside Vcontent luo han guo sweetener.

DETAILED DESCRIPTION OF THE INVENTION

Advantages of the present invention will become more apparent from thedetailed description given hereinafter. However, it should be understoodthat the detailed description and specific examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

The present invention provides a process for purification of high puritymogrosides with high mogroside V content from Siraitia grosvenori fruitextract.

Hereinafter the term “mogrosides” refers to mogrol and oxo-mogrolglycosides, including mogroside IIE, mogroside IIB, mogroside III,mogroside IV, mogroside V, 11-oxo-mogroside V, mogroside VI, siamenosideI, and grosmomoside I.

Hereinafter the term “TM content” means the Total Mogrosides content,and it is calculated as the sum of 4 mogrosides including Mogroside V,11-oxo-Mogroside V, Siamenoside I, and Grosmomoside I.

Hereinafter the term “highly purified” or “high purity” means the TMcontent of at least 70% (w/w) on dry basis.

Hereinafter the term “high mogroside V content” means the mogroside Vcontent of at least 60% (w/w) on dry basis.

Hereinafter the term “low purity” means the TM content of less than 70%(w/w) on dry basis.

Hereinafter the term “low mogroside V content” means mogroside V contentof less than 60% (w/w) on dry basis.

Hereinafter the reported purity levels are determined by HPLC methodwith following parameters. HPLC system—“Agilent 1200 series” (USA)equipped with degasser, quaternary pump, autosampler, thermostattedcolumn compartment and DAD UV detector. Column—“YMC Polyamine II”4.6×150 mm 5 μm (Japan) at 30° C.; mobile phase 70:30 (vol/vol)acetonitrile water at 1 mL/min; detector—UV at 204 nm. Referencestandards of mogroside V, 11-oxo-mogroside V, siamenoside I, andgrosmomoside I were obtained from “Chromadex Inc” (USA).

Hereinafter the term “impurity” means any compound other than mogrosideswhich are present in the mixture at more than 0.0001% (w/w) on drybasis. Non-limiting examples of impurities include proteins, pigments,polysaccharides, aldehydes, unsaturated aldehydes, methyl ketones, butylcrotonate, phenolic compounds as well as other non-mogroside compoundswhich may affect the organoleptic characteristics of luo han guosweetener.

The process of purification of mogrosides of the present invention isapplicable for any low purity mogrosides mixture with the TM content ofless than 70% w/w on dry basis.

The present invention also provides a column system that can be used forthe process of purification of mogrosides of the present invention. Thecolumn system is with following parameters. The column system comprisesa plurality of columns that are connected in either consecutive (serial)or parallel manner, where the plurality of columns are of same ordifferent volumes packed with a sorbent with different affinity toimpurities and steviol glycosides. When the column system is used inparallel connection mode, the inlet of each column may connect to aseparate feed source while the outlet to a separate receiver. Thefollowing description will highlight the mode of column connectionspecific for each particular stage. Within the same stage the system mayfunction as an entity of several parallel and serial connected columngroups and separate columns. The number of columns is 3-15. The ratio ofvolumes of first column to volume of second column is 1:1 to 1:10. Theratio of volumes of last column to volume of previous column is 3:1 to1:10. The columns are packed with sorbent up to 75-100% of their totalvolume. The columns are maintained at temperature 5-80° C.

The alcohol is selected from the group consisting of alkanols, moreparticularly methanol, ethanol, n-propanol, 2-propanol, 1-butanol,2-butanol.

The sorbent is any macroporous polymeric adsorption resin capable ofadsorbing mogrosides, such as Amberlite® XAD series (Rohm and Haas),Diaion® HP series (Mitsubishi Chemical Corp), Sepabeads® SP series(Mitsubishi Chemical Corp), Cangzhou Yuanwei YWD series (CangzhouYuanwei Chemical Co. Ltd., China) or equivalent.

The process of the present invention comprises a few stages. In oneembodiment, the first stage comprises passing an aqueous or aqueousalcoholic solution of low purity mogrosides mixture through aconsecutively connected column system. The water to alcohol ratio(vol/vol) in the aqueous alcoholic solution is 99.9:0.1 to 60:40. As aresult, the impurities and different mogrosides are retained indifferent sections of the column system. Impurities with high affinityare mostly retained in the first column, and low affinity impurities aremostly retained in the last column, while mogrosides are mostly retainedin the middle columns of the column system. Lower molecular weightmogrosides, particularly siamenoside I and grosmomoside I, partiallyseparated from higher molecular weight mogrosides, are retained athigher amounts in the first few columns of the middle columns of thecolumn system, whereas higher molecular weight mogrosides, particularlymogroside V, are retained in the last few columns of the middle columnsof the column system.

In one embodiment, the column system is composed of 6 columns that areconnected in a consecutive manner. In the first stage, an aqueousalcoholic solution comprising a low purity mogrosides mixture with34.73% (w/w on dry basis) comprising Mogroside V 19.47%,11-oxo-Mogroside V 5.60%, Siamenoside I 5.20%, and Grosmomoside I 4.46%was passed through the column system; then each column was eluted withpure alcohol for total elution in order to check the retentions ofmogrosides by each column. The results of the total elution from thefirst stage are summarized in Table 2.

TABLE 2 Mogrosides distribution after first stage Mogrosides, Columns %w/w dry basis Initial I II III IV V VI Siamenoside I 5.20 2.90 11.308.60 4.10 2.81 1.52 Grosmomoside I 4.46 2.80 9.66 7.50 3.13 2.19 1.4811-oxo-mogroside V 5.60 1.50 2.30 3.30 9.37 11.30 5.80 Mogroside V 19.472.73 2.33 17.94 40.32 44.54 8.96 TM content 34.73 9.93 25.59 37.34 56.9260.84 17.76

In another embodiment of present invention, the second stage of thepurification process comprises washing the columns with retainedmogrosides from the first stage to remove residual impurities whichstill remain after the first stage of separation in order to increasefurther the purity level of final products. In one embodiment, thecolumns are sequentially washed with an acidic aqueous or aqueousalcoholic solution, a basic aqueous or aqueous alcoholic solution, andan aqueous alcoholic solution. The water to alcohol ratio (vol/vol) inaqueous alcoholic solution is 99.9:0.1 to 60:40. The removal ofimpurities is carried out either from each column separately (parallelconnection) or more than one consecutively (serial) connected columngroups. Similar to the first stage, each column was eluted with purealcohol for total elution in order to check the retentions of mogrosidesby each column. The results of the total elution from the second stagewith the usage of the same low purity mogrosides mixture and the same 6column system as in the first stage are summarized in Table 2.

TABLE 3 Mogrosides distribution after second stage Mogrosides, Columns %w/w dry basis Initial I II III IV V VI Siamenoside I 5.20 5.28 20.4114.77 6.26 4.03 2.56 Grosmomoside I 4.46 5.09 17.45 12.88 4.73 3.16 2.5411-oxo-mogroside V 5.60 2.73 4.15 5.67 14.34 16.25 9.85 Mogroside V19.47 4.96 4.21 30.81 61.52 64.05 15.22 TM content 34.73 18.06 46.2364.13 86.85 87.49 30.16

In one embodiment of present invention, the third stage of thepurification process comprises eluting the middle section columns (e.g.,columns IV and V) with an aqueous alcoholic solution, where water toalcohol ratio (vol/vol) of the aqueous alcoholic solution is 60:40 to0.1:99.9. The elution is carried out either from each column separately(parallel connection) or more than one consecutively (serial) connectedcolumn groups. The elution results in high purity mogrosides.

In one embodiment, the fourth stage of the purification processcomprises removing alcohol from mogrosides eluate obtained after thethird stage and further concentrating and drying the mogrosides eluateto obtain a dried highly purified mixture of mogrosides. Any methodknown to art may be used for ethanol removal, concentration and drying.

In one embodiment, the low purity fractions are combined and subjectedto repeated purification from the first stage to the fourth stage asdescribed above.

In one embodiment, the high purity fractions are combined and dried toproduce high mogroside V content highly purified mixture of mogrosideshaving TM content of 87.18% (w/w on dry basis) comprising Mogroside V62.82%, 11-oxo-Mogroside V 15.32%, Siamenoside I 5.11%, and GrosmomosideI 3.92%.

In one embodiment, the purification process of the present inventionfurther comprises filtering with the usage of ultrafiltration and/ornanofiltration membranes. Membranes with molecular weight cut-off (MWCO)size of 1000, 1500 and 2000 are used. Highly purified mogroside mixturecomprising Mogroside V 62.82%, 11-oxo-Mogroside V 15.32%, Siamenoside I5.11%, and Grosmomoside I 3.92% was dissolved in aqueous alcoholsolution with alcohol content of 0-100% (vol/vol) preferably 0-50%, tomake a solution with total solids content of 0.1-50% (wt/vol),preferably 0.5-10%. The resulted solution was consecutively passedthrough ultrafiltration and/or nanofiltration membranes with MWCO 1000,1500, 2000 and 2500. A stirred cell membrane system from SterlitechCorp. (USA) was used for this purpose. Anyway any suitable filtrationsystem known to art may be used for this purpose. Non-limiting examplesof membrane manufacturers are Koch Membrane Systems Inc. (USA),GE-Osmonics (USA), Alfa Laval (Sweden). Flat sheet, hollow fiber, spiraland other membranes may be used. Diafiltration was used to increasemembrane filtration process efficiency. Depending on membrane size theretentate or permeate contained the main amount of mogrosides. Aftereach membrane treatment the mogroside containing fraction (retentate orpermeate) was concentrated or diluted again till total solids content0.1-50% (wt/vol) preferably 0.5-10% and passed through the nextmembrane. The solution was passed through increasing membrane sizes(from MWCO 1000 to 2500). Anyway other order of membrane is alsopossible. After membrane treatment highly purified mogroside mixture wasobtained comprising Mogroside V 67.13%, 11-oxo-Mogroside V 16.31%,Siamenoside I 5.51%, and Grosmomoside I 4.10% (dry basis).

The high purity mogrosides can be used either alone or in combinationwith other high intensity sweeteners in food, beverage, pharmaceuticalcomposition, tobacco, nutraceutical, oral hygienic composition, orcosmetic. The other high intensity sweeteners include steviol glycosidesincluding a purified sweet steviol glycoside mixture, stevioside,rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,rebaudioside E, rebaudioside F, dulcoside A, dulcoside B, rubusoside,and stevia; siamenoside; mogroside IV; mogroside V; Luo Han Guosweetener; monatin and its salts (monatin SS, RR, RS, SR); glycyrrhizicacid and its salts; curculin; thaumatin; monellin; mabinlin; brazzein;hernandulcin; phyllodulcin; glycyphyllin; phloridzin; trilobatin;baiyunoside; osladin; polypodoside A; pterocaryoside A; pterocaryosideB; mukurozioside; phlomisoside I; periandrin I; abrusoside A;cyclocarioside I; and combinations thereof.

The following examples are provided for illustrating the process andcolumn system of the present invention.

Example 1

Purification of High Purity Mogrosides

100 g of luo han guo extract (w/w on dry basis) comprising Mogroside V19.47%, 11-oxo-Mogroside V 5.60%, Siamenoside I 5.20%, and GrosmomosideI 4.46% (TM content 34.73%) was dissolved in 2.4 liters of deionizedwater. The solution was passed through 6 consecutively connected 200 mLcolumns packed with Diaion® HP (Mitsubishi Chemical Corp) macroporousadsorbent. 2.4 L deionized water was subsequently passed through thesystem. Further the columns were separately washed with 2 volumes of0.5% HCl, then with water until neutral pH, then with 0.5% NaOH andagain with water until neutral pH of washing waters is achieved. Thenthe columns were separately washed with 10% Ethanol. Then the columnswere eluted with 50% Ethanol. Columns 4 and 5 were connectedconsecutively. The eluate of column 4 and 5 was evaporated under vacuumfor ethanol removal. Obtained aqueous solution was spray dried to yield31 g of highly purified mogroside mixture comprising Mogroside V 62.82%,11-oxo-Mogroside V 15.32%, Siamenoside I 5.11%, and Grosmomoside I 3.92%(on dry basis).

Example 2

Membrane Purification.

100 g of high purity mogrosides prepared according to EXAMPLE 1comprising Mogroside V 62.82%, 11-oxo-Mogroside V 15.32%, Siamenoside I5.11%, and Grosmomoside I 3.92% (on dry basis) was dissolved in 2.4liters of deionized water. The obtained solution was further passedthrough stirred cell membrane system (Sterlitech Corp., USA). Thesolution was passed through GE Osmonics 2500 MWCO membrane. The permeatewhich contained the mogrosides was concentrated to 5% solids and furtherpassed through 1000MWCO membrane. The retentate which contained themogrosides was concentrate and dried to yield 90 g powder containingMogroside V 67.13%, 11-oxo-Mogroside V 16.31%, Siamenoside I 5.51%, andGrosmomoside I 4.10% (dry basis).

While the present invention has been described with reference toparticular embodiments, it will be understood that the embodiments areillustrative and that the invention scope is not so limited. Alternativeembodiments of the present invention will become apparent to thosehaving ordinary skill in the art to which the present inventionpertains. Such alternate embodiments are considered to be encompassedwithin the spirit and scope of the present invention. Accordingly, thescope of the present invention is described by the appended claims andis supported by the foregoing description.

1-4. (canceled)
 5. A high purity mogrosides mixture comprising:Mogroside V in the range of 60-70 wt %; 11-oxo-Mogroside V in the rangeof 10-20 wt %; Siamenoside I in the range of 3-7 wt %; and GrosmomosideI in the range of 2-6 wt %.
 6. A sweetener mixture comprising the highpurity mogrosides mixture of claim 5, wherein the high purity mogrosidesmixture is blended with another high intensity sweetener.
 7. Thesweetener mixture comprising the high purity mogrosides mixture of claim6, wherein the another high intensity sweetener is selected from thegroup consisting of steviol glycosides including a purified sweetsteviol glycoside mixture, stevioside, rebaudioside A, rebaudioside B,rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F,dulcoside A, dulcoside B, rubusoside, and stevia; siamenoside; mogrosideIV; mogroside V; Luo Han Guo sweetener; monatin and its salts (monatinSS, RR, RS, SR); glycyrrhizic acid and its salts; curculin; thaumatin;monellin; mabinlin; brazzein; hernandulcin; phyllodulcin; glycyphyllin;phloridzin; trilobatin; baiyunoside; osladin; polypodoside A;pterocaryoside A; pterocaryoside B; mukurozioside; phlomisoside I;periandrin I; abrusoside A; cyclocarioside I; and combinations thereof.8. A product comprising the high purity mogrosides mixture of claim 6.9. The product of claim 8, wherein the product is selected from thegroup consisting of food, beverage, pharmaceutical composition, tobacco,nutraceutical, oral hygienic composition, or cosmetic.